During nodulation of alfalfa (Medicago sativa) by Sinorhizobium meliloti, the presence of a bacterial exopolysaccharide, succinoglycan, is necessary for the bacterial cells to enter alfalfa root hair cells through a process that resembles the endocytosis of bacterial cells by animal cells. The production of succinoglycan by S. meliloti appears to be up regulated before bacterial cells enter the alfalfa root hairs and then down regulated after the cells reach targeted plant cells in the root nodules. Succinoglycan production is inhibited by the presence of ammonia, which is the end product of this symbiosis. This is consistent with the possibility that succinoglycan participates in the signal exchanges between S. meliloti and alfalfa, which result in the formation of infection threads. Three S. meliloti genes, exoS, chvI, and exoR, are involved in regulating succinoglycan production by controlling the expression of the exo genes, the succinoglycan biosynthesis genes. ExoR mediates the ammonia regulation of succinoglycan production. ExoS, a membrane protein, and ChvI are part of a two-component regulatory system. The target of this regulation is the promoter region of the exoY gene that encodes galactose transferase, which carries out the first step of succinoglycan symbiosis. What remain unknown is the many genes that comprise the ExoS/ChvI and ExoR (ammonia sensing) pathways, the relationship between the pathways, and the exoY promoter(s) that are controlled by the systems. Proposed experiments are focusing on identifying the regulators involved and understanding their roles in nodulation. 1) The exoYpromoters will be fully characterized and used as the framework to identify regulators that interacts with the promoters. This includes mapping the promoter region, determining the number and start sites of the transcripts, determining which exoYFQ promoter is up-regulated, and identifying and isolating proteins that interact with the promoter. 2) The genes down stream of the ExoS/ChvI system in regulating the exo gene expression will be identified through genetic mutagenesis and screening, and though genomic analysis using microarrays. 3) The genes in the ammonia sensing pathway will be identified by screening the genes involved in nitrogen metabolism and ammonia uptake, and by identifying the genes down stream of the exoR gene using genetic and genomic approaches. The newly identified genes will be characterized for their roles in regulating succinoglycan production and nodulation in general. These findings will not only improved the understanding the S. meliloti-alfalfa symbiosis and other bacterial-legume symbiosis, but also provide insights into microbe host interactions in general.